Summary How regenerating axons navigate to their original targets is a central question in the field of axon regeneration. Although several of the mechanisms that guide axons to their targets during embryonic development are well understood, mechanisms governing axon guidance during regeneration are less understood. To elucidate the cellular and molecular mechanisms that guide regenerating axons toward their original targets, we study target-specific regeneration of spinal motor nerves in larval zebrafish. Each nerve is composed of a ventral and a dorsal branch that diverge at a stereotyped branch-point or choice-point. Following laser-mediated nerve transection, axons of the dorsal branch select their original, dorsal trajectory with high fidelity (>70%). We have recently shown that the extracellular matrix component collagen-4-alpha-5 (col4a5) is required for dorsal target selectivity: regenerating dorsal axons in col4a5 mutants frequently (57%) fail to regenerate towards their original targets, and instead grow along ectopic trajectories. After nerve injury, denervated Schwann cells distal to the lesion site express high levels of both col4a5 and the repulsive guidance cue slit1a (Isaacman-Beck et al, Neuron, 2015). This result suggests a role for Slit-Robo signaling in dorsal target selection after nerve injury, so I examined mutants for the Slit receptor roundabout2 (robo2). Regenerating dorsal axons in robo2 mutants frequently (45%) fail to regenerate towards their original targets, and instead extend along ectopic trajectories. In contrast, robo2 is dispensable for ventral axon regeneration. Therefore, I next asked whether robo2 is sufficient to promote dorsal (as opposed to ventral) target selection by regenerating axons. Following nerve transection, I find that ventral axons in which robo2 is stochastically force-expressed frequently (~50%) regenerate along the dorsal path. These preliminary findings support a model in which col4a5-expressing Schwann cells repel robo2-expressing dorsal axons, directing them toward dorsal targets in regeneration. Here I propose to test this model by determining whether the canonical Robo-binding partners, Slits, are required for dorsal axon guidance in regeneration (Aim 1a) and whether non-canonical robo2 ligands may be guiding regenerating axons (Aim 1b). Finally, I will test whether col4a5 and robo2 function in the same genetic pathway to guide regenerating dorsal axons (Aim 2). The proposed experiments will enhance our understanding of robo2 signaling and will elucidate regeneration-specific axon guidance mechanisms. Understanding guidance mechanisms employed by regenerating axons is crucial for designing therapeutics for functional recovery from peripheral nerve injury. 1